The present invention relates to a detection system and method for measuring fluorescently labeled analytes by their interactions with particles encoded with fluorescent labels, and further relates to fluorescent label compositions for coding polymeric microbeads or particles.
Polymeric beads are useful analytical tools for detecting and measuring various analytes especially when combined with flow cytometry systems and methods. The term polymeric beads is referred to in the art and used hereinafter interchangeably as beads, particles, microbeads, microparticles, and microspheres. Analytes of interest are often bound to a particle and identified by a corresponding characteristic of the particle such as size, magnetism, and spectroscopic properties including absorbance, light scatter, color, and fluorescence at one or more wavelengths.
For example, prior art patents describe the use of particle size or color as parameters for distinguishing between subpopulations of particles. A disadvantage of employing size or color as a distinguishing markers is that these systems permit the labeling of only a few distinct subpopulations of particles. Employing additives of differing absorbance to mark different particle subpopulations has also been described. A disadvantage of absorbance markers is that absorbance in a particle is difficult to measure and is not a particularly sensitive method of detection.
Fluorescence characteristics of particles or cells has been described in a variety of analytical systems including fluorescence microscopes, flow cytometers and image microscopes for analyte identification. Fluorescent labels are desirable markers for coding particles and have been described in a variety of different approaches including employing single and multiple fluorescers as labels. The use of fluorescent labels as markers in flow cytometry systems is described, for example, in U.S. Pat. Nos. 4,745,285; 5,028,545; 5,682,038; and 5,880,474, all of which are incorporated herein by reference. However, there are several distinct disadvantages to prior systems.
As with particle size, the use of a single fluorescent marker by itself enables labeling of only a few distinct subpopulations of particles. Prior systems employing multiple fluorescent labels can be disadvantaged when separate space is not reserved for the emission spectra for the analyte of interest. Overlapping emission spectra between an analyte and a fluorochrome can hinder detection and quantification of the analyte in these systems.
Many naturally occurring samples and materials for instrument construction contain materials, which fluoresce in the UV or the short-wavelength end of the visible spectrum. These extraneous sources of fluorescence interfere with particle detection and with accurate detection and quantification of analytes by fluorescent labeling.
When multiple fluorescent labels are used, the multiple fluorescent emission spectra may be indistinct due to dye to dye interactions, overlapping spectra, and non-Gaussian emission profiles. Indistinct emission spectra make accurate identification and quantification between multiple subpopulations of particles difficult. Interaction between multiple fluorescent labels limits the number of distinguishable particle species and interaction between the fluorescent labels and fluorescent analytical dyes limits the quantitative detection capabilities of the device. Complex signal processing devices must be employed to compensate for the indistinct spectra, adding to the cost of the detection system.
Prior analytical detection systems employing particle technology suffer from one or more of the following disadvantages: 1) limited accuracy; 2) limited sensitivity; 3) inadequate numbers of labels for the multitude of analytes to be detected; 4) expensive equipment; and 5) time consuming multiple reaction steps. A need, therefore, exists for an analytical detection system employing particle technology that can distinguish between multiple subpopulations of particles in a cost and time efficient manner while simultaneously accurately identifying and quantifying multiple analytes.